Project summary A cell is like a city, with an organized yet dynamic infrastructure grouped into specialties. For the last 25 years, my lab has investigated how the simplest cells?bacteria?organize themselves and divide to make progeny cells. We mainly focus on how bacteria such as E. coli achieve the daunting task of splitting themselves in two at just the right time (once their genetic material is duplicated) and place (exactly in the middle) every 20 minutes without making errors. The keys to this success are ancient and universal versions of protein polymers of actin (FtsA) and tubulin (FtsZ), which our lab visualized for the first time in living bacteria over 20 years ago. Today, we use state of the art super-resolution imaging, combined with molecular genetics, protein biochemistry, interaction studies, and in vitro reconstitution, to gain more detailed insights into the structure and regulation of these cytoskeletal polymers and their associated proteins, which comprise the dynamic membrane-associated protein nanomachine (divisome) that divides bacterial cells. Thanks in part to our characterization of bypass suppressors of essential divisome proteins, it is now becoming clear that the divisome is highly flexible, and can remodel itself in response to various inputs and perturbations. Despite impressive contributions by many labs, there is much to be learned about overall divisome structure, the interchangeability of its parts, and how it remodels in response to temporal and environmental cues. We will address these fundamental questions by (1) obtaining more high-resolution information about protein-protein contacts during cytokinesis by combining biophysical, cytological, and genetic approaches; (2) investigating the role of oligomeric state of FtsZ and FtsA in divisome function and regulation, using super-resolution microscopy of whole cells and reconstituted biomimetic protein-membrane systems; (3) taking advantage of the diversity of divisome proteins from other model bacterial species to distinguish between common and specialized mechanisms; (4) understanding the interplay between the divisome and other large-scale cellular processes such as cell wall biosynthesis. We will leverage these approaches by continuing our collaborations with several close colleagues who have complementary interdisciplinary expertise. Our ongoing investigation of how the simplest cells divide should pave the way for an unprecedented understanding of how an entire cell functions and reproduces. Having an accurate map of that city-cell's dynamic infrastructure will allow predictions to be made about how it works, and how to disrupt it.